Abstract

Many steps of viral replication are dependent on the interaction of viral proteins with host cell components. To identify rhinovirus proteins involved in such interactions, human rhinovirus 39 (HRV39), a virus unable to replicate in mouse cells, was adapted to efficient growth in mouse cells producing the viral receptor ICAM-1 (ICAM-L cells). Amino acid changes were identified in the 2B and 3A proteins of the adapted virus, RV39/L. Changes in 2B were sufficient to permit viral growth in mouse cells; however, changes in both 2B and 3A were required for maximal viral RNA synthesis in mouse cells. Examination of infected HeLa cells by electron microscopy demonstrated that human rhinoviruses induced the formation of cytoplasmic membranous vesicles, similar to those observed in cells infected with other picornaviruses. Vesicles were also observed in the cytoplasm of HRV39-infected mouse cells despite the absence of viral RNA replication. Synthesis of picornaviral nonstructural proteins 2C, 2BC, and 3A is known to be required for formation of membranous vesicles. We suggest that productive HRV39 infection is blocked in ICAM-L cells at a step posttranslation and prior to the formation of a functional replication complex. The observation that changes in HRV39 2B and 3A proteins lead to viral growth in mouse cells suggests that one or both of these proteins interact with host cell proteins to promote viral replication.

Growth of HRV39 and RV39/L in cultured cells. Wild-type HRV39 or RV39/L was used to infect (A) ICAM-L cells or (B) HeLa R19 cells at a multiplicity of infection of 10. Infections were halted at the indicated times postinfection, and viral titers were determined by plaque assay.

Multiple alignment of picornaviral 2B and 3A proteins. The boxed regions in both proteins represent hydrophobic domains. Dashed boxes in 3A correspond to the 3A homodimer interface. Amino acid differences between HRV39 and RV39/L proteins are indicated by underlined residues in the HRV39 sequence. The amino acid changes in RV39/L are shown above the HRV39 residue.

Schematic for generation of viral transcripts containing mouse-adapted amino acid changes in either the 2B or 3A protein. A DNA copy of the HRV39 and RV39/L viral genomes was cloned into pACYC177. The plasmids were cleaved with BsiEI and SacI or BsiEI and SphI, and DNA fragments containing (i) the T7 promoter and the 5′ half of the viral cDNA or (ii) the 3′ half of the viral cDNA and the poly(A) sequence were purified. Ligations of the 5′ half of HRV39 DNA and the 3′ half of RV39/L DNA or the 5′ half of HRV39/L DNA and the 3′ half of HRV39 DNA resulted in full-length DNAs with changes in either 2B or 3A. RNA transcripts were generated by in vitro transcription, and the RNA was introduced into cells by transfection to produce virus.

Growth of HRV39, RV39/L, RV39/IGE, and RV39/RM in cultured cells. Infections were carried out in (A) ICAM-L cells or (B) HeLa R19 cells at a multiplicity of infection of 10. Infections were halted at the indicated times postinfection, and viral titers were determined by plaque assay.

Positive-strand viral RNA production in infected cells. Infections were carried out in (A) ICAM-L cells or (B) HeLa R19 cells at a multiplicity of infection of 10. At 0, 6, and 12 h postinfection, total RNA was isolated from infected cells and transferred onto a nitrocellulose membrane. A radiolabeled RNA hybridization probe complementary to positive-strand viral RNA was used to measure levels of viral RNA present at various times postinfection. The amount of hybridized RNA probe was determined with a PhosphorImager, analyzed using ImageQuant software, and reported as optical density.

Formation of membranous vesicles in infected cells visualized by electron microscopy. HeLa R19 cells are (A) uninfected or infected with (C and G) HRV39 or (D and H) RV39/L. ICAM-L cells were (B) uninfected or infected with (E and I) HRV39 or (F and J) RV39/L.